We developed systems to investigate hepatocyte lineage life history dynamics in vivo. We propose to define the factors that determine hepatocyte lineage birth-rates and longevities, and to describe their dynamic responses to hepatic stresses in aging. In this collaborative proposal, empirical in vivo studies are combined with mathematical modeling and simulation to test effects of extrinsic and intrinsic factors on hepatocellular lineage dynamics and how these change as a part of the aging process in a genetically tractable animal model. What is known. Unlike most differentiated cell types, hepatocytes can proliferate. When normal liver cells are transplanted into mice having a genetic defect that autonomously compromises the endogenous hepatocytes, the grafted cells can complete >18 consecutive replicative cycles, resulting in full replacement of the endogenous hepatocytes and reconstitution of the liver with healthy cells. Using serial reconstitution through 7 consecutive recipient mice, a classic study showed that adult wild-type liver cells could undergo an average of at least 69 consecutive divisions 1. Thus, liver cells have a nearly unlimited capacity to proliferate 1 and may be """"""""stem cell-like"""""""" in their regenerative immortality 1,2. Hepatocytes are also one of few cell types that undergo endoreplication and acytokinetic mitosis, resulting in polyploid nuclei and bi-nucleate cells, respectively. Most hepatocytes are polyploid and both diploid and polyploid hepatocytes can proliferate. Indeed, another study showed that the most active liver cell types for reconstituting compromised liver are polyploid hepatocytes 3. These background data indicate that: (1) liver cell populations may be infinitely proliferative;(2) hepatocytes are predisposed to becoming polyploid;and (3) polyploid hepatocytes are highly proliferative. Preliminary observations and the problem they reveal. We developed genetic marker systems for """"""""time-stamping"""""""" hepatocyte lineages in vivo 4. In contrast to the prevailing model of the immortal hepatocyte, our systems show that hepatocyte lineages have both a finite half-life and a limited capacity to proliferate. We also developed a flow cytometry-based method of quantifying liver nuclei on the basis of ploidy and found that adult livers between 2- and 12-months of age exhibited nearly invariant ratios of diploid (2N), 4N, and 8N nuclei. Lastly, we developed a novel """"""""ten-day chronometer"""""""" for newly differentiated hepatocyte lineages that allows us to quantitatively assess the contributions of pre-hepatocytic stem cells to liver growth, regeneration, and maintenance 5. Using this chronometer, we found that normal adult liver is continuously gaining new diploid hepatocyte lineages. We believe these replace lineages that die-off due to age or stress. Based on our observations, we suspect that only pre-hepatocyte cell types, not differentiated hepatocytes, have unlimited proliferative potential and that this rare population of cells underlies the proliferative immortality of liver. Our hypothesis and how we will test it. Based on our findings, we hypothesize that hepatocytes have a life history that includes birth from stem cells, age-related deterioration, and death. We predict that hepatic stresses, replication, ploidy, aneuploidy, and time will affect hepatocyte lineage life history dynamics. Moreover, the process of aging of the host animal might influence the life history dynamics of hepatocyte lineages. The quality of either the hepatic stem cells or the """"""""liver niche"""""""" could change as livers age, resulting in differences in hepatocyte lineage birth rates, longevities, proliferative potentials, and stress resistance. To test our hypothesis, we will fulfill four aims: (1) Define birth-rates and longevities of hepatocyte lineages under normal and stressed conditions. (2) Determine what factors limit lineage longevity. (3) Measure the dynamic lineage-aging process in hepatocyte nuclei. (4) Examine how an animal's age and exposure-history affects the life history dynamics of hepatocyte lineages. Implications for human health in aging. Hepatocytes are generally thought to have a stem cell-like capacity to proliferate and regenerate lost or damaged liver tissue. Indeed, the term 'stem cell-like'invokes a level of immortality that has been tested in only a small number of situations. Clearly mouse ES cells, for example those we used to make our various lines of mice having targeted mutations, have been verified through years of culture and mouse-production as being indefinitely self-renewing;but is this true of all organ-specific stem cells? Maintenance of the self-renewing capacity of ES cells in culture requires a very strict environment or """"""""niche"""""""" (e.g., media, supplements, attachment factors, feeder-cells, pH, etc.), so it may be reasonable to predict that the """"""""quality"""""""" of organ-specific stem cells in vivo could also be intimately dependent on the niche that the host-organ provides. This niche could change with an animal's age or exposure history, yet these possibilities have not been previously considered. Here we propose an investigation into the aging process in differentiated hepatocyte lineages and how both this process and the contributions of hepatic stem cells change as animals age. Upon completion of this project, we will have: (a) developed and publicly disseminated novel mouse models for studying aging of hepatocyte lineages;(b) defined rates of birth and death of hepatocyte lineages under normal and several stressed states;(c) characterized the aging process in normal and stressed hepatocyte lineages;and (d) investigated how these processes change as a function of aging and exposure history of the host animal.

Public Health Relevance

Mice, like humans and likely all mammals, undergo a process of age-dependent deterioration, or aging, which is associated with accumulation of molecular damage and often with a reduced capacity to renew or regenerate existing tissue. Many adult tissues have tissue-specific stem cells, which are thought to possibly play a role in rejuvenating the tissue and thereby countering or delaying the aging process;however in most cases, these cells are rare and their activities are poorly studied. Liver is a large metabolically active organ that plays numerous crucial roles in organismal physiology. The organ is composed predominantly of a single cell type, the differentiated hepatocyte, which is responsible for most of the liver's functions. Unlike most differentiated cells in the body, hepatocytes can proliferate and self- renew. Indeed, currently accepted models suggest that hepatocytes are immortally self-renewing, and therefore play a stem cell-like role in organ maintenance. Curiously, it is known that liver does contain a small population of hepatic stem cells;however, no role for these cells in normal liver physiology or maintenance has yet been established. It is our belief that some of these properties of hepatocytes and hepatic stem cells are inaccurate, and stem from a lack of suitably sensitive means of assessing either the immortality of hepatocyte lineages or the contributions of hepatic stem cells. We have developed innovative and highly sensitive fluorescent lineage-marker systems for measuring rates of birth of new hepatocyte lineages from pre-hepatocytes and for following hepatocyte lineages and quantitatively assessing their decay in vivo. As such, these systems allow very sensitive measures of processes that had previously been undetectable, and they are revealing some unexpected properties of hepatocytes and hepatic stem cells. Our preliminary studies show that pre-hepatocytes, most likely the hepatic stem cells, play a constant role in generating new lineages of proliferative hepatocytes, and that these lineages decay with time. Thus, we have found that hepatocyte lineages have a life history that includes birth from a pre-hepatocyte stem cell population, aging, and death, which is constantly occurring in resting liver. Moreover, we have found that some changes in the physiological state of the liver can alter the rate of birth of new hepatocyte lineages. We believe that this birth of new hepatocyte lineages is of crucial importance for long-term maintenance of the liver. In the proposed project, we test how this contribution changes as an animal ages, how the longevity of hepatocyte lineages change as an animal ages, and whether the hepatotoxic exposure or hepatic stress histories of an animal alters hepatocyte lineage life history dynamics. The proposed study integrates descriptive studies on hepatocyte lineage dynamics in aging animals with mechanistic studies on the aging process and with computational modeling of the cellular dynamics in young and aged liver. This is a collaborative proposal that integrates the expertise of a mouse molecular geneticist/cell biologist with that of a bio-mathematician to provide an accurate, quantitative, and predictive analysis of the aging process in liver. A plan for integrating the empirical and computational subgroups of this project is provided. Novel mouse systems are developed within this proposal and a resource-sharing plan is included for these are to be made freely available to the international research community for continued investigations along these or other lines.

National Institute of Health (NIH)
National Institute on Aging (NIA)
Research Project (R01)
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Special Emphasis Panel (ZAG1-ZIJ-2 (M1))
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Murthy, Mahadev
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Montana State University - Bozeman
Veterinary Sciences
Schools of Earth Sciences/Natur
United States
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